Studies on betalain phytochemistry by means of ion-pair countercurrent chromatography

Abstract

Betalains display color from yellow-orange to red-purple within few species of Chenopodiaceae family. The specific structures and consistencies upon pH are the most applicative features of betalains. Only several betalains are heavily understood and available as references due to their instabilities and rare appearances in only several edible crops. That requires robust techniques for large scale production and investigation of new betalainic sources. Countercurrent chromatography and centrifugal partition chromatography are preparative purification techniques based on continuous liquid-liquid extraction and have been applied on natural pigments such as anthocyanins and betalains. This work added to a few studies on betalains from some highly pigmented cultivars as Opuntia, Atriplex and Hylocereus; which results confirmed the feasibility of preparative isolation of pigments using CCC. The application of ion pair CCC on Opuntia pigments showed comparable distribution values between analytical and preparative high performance spectrum. Similar results were seen in the comparison between CCC and CPC studies, indicating the scale-up possibility of these methods. The Opuntia metabolites were investigated next to pigments, from which betalains and flavonoid glycosides were isolated from other cyclicpeptides. This work also examined the heat stability of Opuntia pigments that the un-resolvable of intact pigments from their thermal artifacts were witnessed. This study developed a large lab scale pre-purification process for complex extracts of betalain plants, using highly available and bio-degraded chitin. This approach including simple adsorption and desorption steps based on strong pigments binding capacity of the chitin. The procedure was optimized and proved its capacity to concentrate the minor pigments from heterogeneous betalain crude extracts of red beet and Opuntia. This enrichment approach was then applied to Atriplex and Hylocereus in larger scale prior to CCC separation. The combination of this chitin process with CCC using ion pair solvent system TBME/n-BuOH/ACN/water TFA monitored by ESI-MS/MS profile could fractionate the pigments of Atriplex from their flavonoids, and isolated high purity celosianin II for NMR structural identifications. The un-resolve of dragon fruits pigments indicated its high polarities and required stronger ion pair reagents. This result confirmed the indispensable of ion pair reagents within CCC separation of betalains.